Hyperactivation of toll-like receptors (TLRs) leads to inflammatory conditions and disease states, both pathogenic and sterile, such as sepsis, autoimmune disorders and ischemia reperfusion injury during organ transplantation. This proposal is centered on the use of a newly developed liposomal spherical nucleic acid nanoparticle (LSNA) carrying TLR antagonists to probe the molecular mechanism of immune activation by TLRs and to advance the development of a novel therapeutic platform for potential treatment of inflammation. LSNAs are novel nanomaterials that withstand degradation, lead to higher receptor binding affinities and have enhanced potency due to their 3D architecture and oligonucleotide arrangement around a lipid core that confers enhanced biological properties beyond their individual components alone. The elements of the LSNA nanoparticle, specifically the chemistry governing the liposomal core and the oligonucleotide shell, provide a platform for integrating target specificity into the design of the delivery system and payload delivery that permits potent inhibition of TLR activation. This design allows for inhibition of multiple, yet distinct, receptor subtypes. This proposal investigates the biological function of a dual TLR-inhibitory LSNA, which has been previously synthesized and validated, to inhibit distinct TLR ligands that differ in cellular location, but are jointly involved in propagating injury from tissu ischemia and reperfusion. The central goal of this transdisciplinary and collaborative project is t explore the chemical properties of LSNAs that govern biological efficacy and specificity in modulating downstream TLR signaling pathways. Aim 1 probes the oligonucleotide backbone chemistry and specific sequence alterations with the goal of correlating chemical composition with potency of TLR inhibition and immune activation using cellular tools, such as engineered cell lines and primary human lymphocytes. Aim 2 investigates novel methods to minimize ischemia reperfusion injury using LSNAs in an in vivo model of organ transplantation. The use of targeting nanoparticles with the potential to selectively inhibit more than one receptor is an innovative approach because TLR co-stimulation and pathway crosstalk is implicated in multiple diseases and pathologic states. If effective, there are widespread possibilities to applying this nanoparticle for therapeutic applications to other inflammatory diseases in which TLR hyperactivity has been implicated.